Muscles, Ligaments and Tendons Journal 2013; 3 (4): 254-266254
Mitohormesis in muscle cells: a morphological,molecular, and proteomic approach
Elena Barbieri
Piero Sestili
Luciana Vallorani
Michele Guescini
Cinzia Calcabrini
Anna Maria Gioacchini
Giosuè Annibalini
Francesco Lucertini
Giovanni Piccoli
Vilberto Stocchi
Department of Biomolecular Sciences, Division of Ex-
ercise and Health Sciences, University Carlo Bo,
Urbino, Italy
Corresponding author:
Elena Barbieri
Department of Biomolecular Sciences, Division of Ex-
ercise and Health Sciences
University Carlo Bo
Via I Maggetti, 26
61029 Urbino, Italy
E-mail: [email protected]
Summary
Low-level oxidative stress induces an adaptive re-
sponse commonly defined as hormesis; this type
of stress is often related to reactive oxygen
species (ROS) originating from the mitochondrial
respiratory chain (mitochondrial hormesis or mito-
hormesis). The accumulation of transient low dos-
es of ROS either through chronic physical activity
or caloric restriction influences signaling from the
mitochondrial compartment to the cell, reduces
glucose metabolism, induces mitochondrial me-
tabolism, increases stress resistance and ulti-
mately, increases lifespan. Mitochondrial forma-
tion of presumably harmful levels (chronic and/or
excessive) of ROS within skeletal muscle has
been observed in insulin resistance of obese sub-
jects, type 2 diabetes mellitus, as well as in im-
paired muscle function associated with normal ag-
ing. Advances in mitochondrial bioimaging com-
bined with mitochondrial biochemistry and pro-
teome research have broadened our knowledge of
specific cellular signaling and other related func-
tions of the mitochondrial behavior. In this review,
we describe mitochondrial remodeling in re-
sponse to different degrees of oxidative insults in-
duced in vitro in myocytes and in vivo in skeletal
muscle, focusing on the potential application of a
combined morphological and biochemical ap-
proach. The use of such technologies could yield
benefits for our overall understanding of physiolo-
gy for biotechnological research related to drug
design, physical activity prescription and signifi-
cant lifestyle changes.
KEY WORDS: mitochondria, skeletal muscle, ROS,
hormesis.
Introduction
Mitochondria are dynamically involved in several
muscle cellular activities including signaling, prolifera-
tion, differentiation, autophagy and death. Skeletal
muscle cells, as well as the metabolically active neu-
rons, accumulate the highest number of mitochon-
dria. The dynamics, size, number and location of mi-
tochondria in muscle cells vary significantly according
to the cellular conditions (muscle fiber types, differen-
tiation level, training, nutritional state) and energetic
needs (contraction, steady-state, recovery). For in-
stance, mitochondria accumulate predominantly at
high energy demanding sites, close to calcium re-
lease units in striated muscle, providing a physical
basis for localized Sarcoplasmic Reticulum - mito-
chondrial Ca2+ signaling and suggesting that mito-
chondria participate in Ca2+ cycling at the triad level1.
Recently, the identity of mitochondrial Ca2+ trans-
porters has been revealed, opening new perspectives
for investigation and molecular intervention2. Given
the role of mitochondria in a wide range of cellular
processes, it is not surprising that mitochondrial dam-
age has been implicated in the pathogenesis of sev-
eral muscle as well as neurodegenerative or chronic
diseases3,4. Mitochondria are generally considered as
the main source of reactive oxygen species (ROS) in
skeletal muscle cells. It is known that 2-5% of the to-
tal oxygen consumed by mitochondria may undergo
one electron reduction with the generation of super-
oxide. Complexes I and III of the electron transport
chain seem to be the main sites of mitochondrial su-
peroxide generation5,6. Several physiological and
pathological conditions may result in ROS production
by mitochondria. For example, high intense contrac-
tile activity, disuse muscle atrophy or inflammation,
increased mitochondrial ROS generation7. Although
the excessive ROS production is associated with the
aetiopathology of several human diseases, low levels
of ROS are important mediators for a variety of cellu-
Review article
Muscles, Ligaments and Tendons Journal 2013; 3 (4): 254-266 255
Mitohormesis in muscle cells: a morphological, molecular, and proteomic approach
tive stress, apoptosis, and Ca2+ concentrations10.
Most of the mitochondrial dyes are taken up by the mi-
tochondrial membrane potential. Such fluorescent dye
markers may stain the structure of whole organelles,
as well as mitochondrial parameters, such as redox
potential, Ca2+ and Cl- levels, and H+ relevant to the
organelle functionality10. The dye fluorescence varia-
tions may depend on the surrounding environment
and can also be used to measure the mitochondrial
membrane potential, mass/volume, or oxidative state
conditions in living cells.
Oxidative level inside mitochondria
ROS are very reactive molecules and extremely un-
stable, thus it is impossible to image them directly.
The ROS detection levels have mainly relied on de-
tecting their end products by chemiluminescence or
by fluorescence. These products are usually formed
when specific compounds react with ROS. One of the
most frequently used techniques for detecting intracel-
lular ROS, in particular hydrogen peroxide, depends
on oxidation of the non-fluorescent substrate, such as
2,7-dichlorodihydrofluorescein a green fluorescent
dye11,12. Due to the existence of several substances
that interfere with the formation of 2,7-dichlorofluores-
cein (DCF), this probe, when used in cellular systems,
is more useful as a marker of cellular oxidative
stress11. Cell membranes are permeable to esterified
forms of DCF. These products can easily enter cells
and through deacetylation by esterases, they remain
intra-cellularly trapped. The oxidation rate may be de-
tected by a fluorimeter, fluorescence microscopy or by
flow cytometry. Dihydroethidium (DHE) freely perme-
ates cell membranes; thus, it is used extensively to
monitor superoxide production. It had long been as-
sumed that DHE, after reaction with superoxide an-
ions, forms 2-hydroxyethidium a DNA intercalates
which shows a red fluorescent product12. DHE is pos-
sibly the most specific, it reveals essential superoxide
radicals, is preserved well by cells, and may also
stand mild fixation12. The fluorophore10N-nonyl acri-
dine orange (NAO) tightly binds to the mitochondrial
cardiolipin in mitochondrial membranes in eukaryotes
and bacteria. Cardiolipin content may decrease in ox-
idative stress conditions. The cardiolipin-sensitive
probe NAO may be used to monitor oxidative changes
in mitochondrial lipids and indirectly indicates the mi-
tochondrial peroxidation level13.
Mitochondrial ultrastructure
Electron microscopy has enormously improved its
application during the last few decades and it has
become a powerful tool for studying mitochondria ul-
trastructure and function. Various modifications may
be taken into consideration for sample preparation in
fixation, dehydration, sectioning, inclusions and
staining of the section to maintain the original mor-
phology. In our investigations undifferentiated C2C12
lar processes, including cell adhesion, immune re-
sponse, apoptosis, cell growth and differentiation.
ROS also act as second messengers in intracellular
signaling. In fact, it has become apparent that low
levels of H2O2 may be required for sustaining cellular
functioning and intracellular signaling. Transient in-
creasing levels of ROS play a crucial role in modulat-
ing redox-sensitive gene expression, cell-signaling
pathways and in preserving contractile function8,9.
However, chronic and excessive ROS production
may lead to muscle cell damage and death. The
prevalence of the role of ROS either in promoting
benefits or establishing unfavorable conditions for the
pathogenesis of a several skeletal muscle disorders
or decline in muscle function, depends on the concur-
rence of intrinsic and extrinsic factors. These factors
include the level and duration of ROS targeting mus-
cle cells, the source, the site of ROS generation, the
antioxidant status of target cells, the DNA repair ca-
pacity, the differentiation stage of muscle cells and
the proliferative and myogenic capacity of satellite
cells. Due to their hormetic nature, in muscle tissue
mitochondrial ROS may trigger different signaling
pathways leading to different and diverging respons-
es, from adaptation to cell death. A significant num-
ber of studies describes the role of ROS in triggering
many signaling pathways relevant to skeletal muscle
cells’ homeostasis and adaptation. Herein we will elu-
cidate some of the signaling pathways triggered by
ROS in mitochondrial muscle remodeling and their
physio-pathological implications. Particular attention
will be focused on advances in morphological and
biomolecular approaches combined with mitochondri-
al proteome research in mitochondrial muscle remod-
eling in response to oxidative stress and the direct
link between mitochondrial disruption and several bio-
logical modulations.
Morphological methods
Bio-imaging
Mitochondrial dynamics is a recent designation of mi-
tochondrial behavior. Recent studies have led to re-
newed interest in determining the genes, proteins, and
mechanisms that regulate mitochondrial shape, size,
number, and distribution. Studies on mitochondrial live
remodeling have mostly been performed using fluo-
rescence microscopy10. This technique focuses on the
development of fluorescent microscopy and specific
fluorescent probes to stain mitochondria or specific
mitochondrial molecules. Recent methodological ap-
proaches in fluorescent imaging technologies have
improved our capacity to study both the dynamics and
morphology of mitochondria by following specific
metabolites and ions within mitochondrial sub-com-
partments, such as the membranes and matrix. A high
number of fluorescent probes and dyes are increas-
ingly used to quanti-qualitatively evaluate overall mus-
cle mitochondrial number, membrane potential, oxida-
Muscles, Ligaments and Tendons Journal 2013; 3 (4): 254-266256
E. Barbieri et al.
myoblast and differentiated myotube monolayers
were washed and fixed with 2.5% glutaraldehyde in
0.1 M phosphate buffer for 15 min to maintain their
morphological structure. They were then gently
scraped and centrifuged at 1200 rpm. Cell pellets
were further fixed with glutaraldehyde for 1 h. All
specimens were OsO4 post-fixed, alcohol dehydrat-
ed, and embedded in araldite. Thin sections were
stained with uranyl acetate and lead citrate. Mito-
chondrial density was calculated in 20 different areas
of 10×15 cm at 28000 magnification. Mitochondrial
sizes were evaluated at 28000 magnification using
the Philips CM10 microscope and Megaview soft-
ware system14. Data from our group15 indicate that a
mildly toxic H2O2 treatment during the early stages of
C2C12 myoblast differentiation results in GSH deple-
tion and strongly impairs the differentiative outcome.
Interestingly, H2O2-injured cells showed signs of ex-
tensive mitochondrial degeneration (swelling and dis-
ruption) and lower mitochondrial density, suggesting
that these organelles are specifically targeted by – or
particularly sensitive to – exogenous ROS. Loss of
mitochondria is a clearly detrimental event in a
process typically requiring active mitochondriogene-
sis such as muscle differentiation16. These effects
are visible by the inserts in the TEM micro- graphs of
H2O2-treated cells at both differentiation day (DifD) 3
and 5 of Figure 1.
Mitochondrial subcellular localization
In striated muscle, the subcellular localization of all in-
tracellular mitochondria largely depends on myofiber
organization and plasma membrane invaginations of
the T tubules. Interactions of mitochondria with the
sarco-endoplasmic reticulum (SR/ER) have been re-
ceiving a great deal of attention because of emerging
evidence on the role this site has in cell signaling and
mitochondrial dynamics.
Mitochondria respond to different stimuli through re-
modeling, thus they pass from a dynamic behavior to
a stabilized condition. In fact, they structurally localize
in precise subcellular positions probably anchored
with the face of the junctional SR opposite to that of
the type 1 ryanodine receptor (RyR1) feet. This close
association and tight coupling between the outer mito-
chondrial membrane (OMM) and the calcium releasing
unit (CRU) in muscle is supposed to respond in the lo-
cal domain of calcium signaling and metabolic cou-
pling during normal muscle activity. The contractile ac-
tivity of muscle has to be matched by mitochondrial
ATP generation that is completed, at least in part, by
the propagation of Ca2+ signals from SR to mitochon-
dria. Muscle has a highly geometrical structure, pro-
viding only limited opportunity for mitochondrial dy-
namics and inter-organellar interactions. Mitochondria
are closely tethered to the SR terminal cisternae17, the
RyRs are localized at some distance from mitochon-
dria (about 100nm) as these Ca2+ release channels
are located on the SR surface facing the T tubules18.
Mitochondrial Ca2+ uptake in muscle depends on the
diffusion of Ca2+ released from the SR to the mito-
chondrial surface18.
Triad-targeted mitochondria in adult skeletal muscle
are remarkably stable and immotile. Because CRU–
mitochondrion Ca2+ cross-talk in skeletal muscle oper-
ates via local signaling microdomains, this “privileged”
signaling could best be ensured by restricting mito-
chondria to a location immediately adjacent to sites of
Ca2+ release by a strong structural anchoring mecha-
nism (Fig. 2). Mitofusin 2 is probably the main player
in the formation of such ER/mitochondrial close appo-
sitions. Mitofusin 2 is localized both in the outer mito-
chondrial membrane and on the ER surface19.
Of particular interest is the recent discovery of
SR/ER–mitochondria tethers that are formed by multi-
ple proteins, and local Ca2+ transfer between SR/ER
and mitochondria. Given that the molecular identities
of mitochondrial Ca2+ uniporter (MCU) has been un-
Figure 1. Differentiating C2C12 at SEM and TEM: the effect of oxidative challenge. Micrographs were taken at DifD 3 and 5.
Initial thin myotubes appeared in control cells at DifD3, while an increased number of thicker and longer myotubes were pre-
sent at DifD5. H2O2 induced necrosis at DifD3 and DifD5 (n). The inserts in the TEM micrographs of H2O2-treated cells at
DifD3 and 5 highlight mitochondrial swelling and disruption caused by oxidative stress. Bars: SEM 10 lm; TEM 1 lm. (Adapt-
ed from Sestiliet al.15, with Authors permission).
Muscles, Ligaments and Tendons Journal 2013; 3 (4): 254-266 257
Mitohormesis in muscle cells: a morphological, molecular, and proteomic approach
raveled only very recently2, the information on the tis-
sue expression of these molecules is still fragmentary
and the identity of the specific complex of anchoring
that stabilizes the Mitochondrial-Sarcoplasmic Reticu-
lum is still unknown.
In skeletal muscle, mitochondria occur as two sub-cel-
lular populations identified as subsarcolemmal (SS)
and intermyofibrillar (IMF) mitochondria (Fig. 3). Bio-
chemical and functional differences between IMF and
SS fractions exist, and they probably represent mito-
chondria at different stages of maturation or biogene-
sis. Changes may not only be due to mitochondrial
protein synthesis but also to nuclear-directed protein
synthesis exclusive to each mitochondrial population.
It has been described how acute contractile activity
exerts differential effects on protein turnover in IMF
and SS mitochondria, and it appears that intra-mito-
chondrial protein synthesis does not limit the extent of
chronic contractile activity-induced mitochondrial bio-
genesis20. These mitochondrial subpopulations re-
spond differently to various physiological perturba-
tions. For example, SS mitochondria appear to be
more malleable in response to exercise and pharma-
cological interventions. It seems possible that SS mi-
tochondria possess more sensitive transcriptional con-
trol of their mitochondrial genome, although this con-
cept has yet to be investigated.
Figure 2. Mitochondria and CRUs are close to each other and connected by small electron dense strands, or tethers. (A) In
adult muscle fibers, triads (small black arrows) are located in proximity to the sarcomere A-I band junction. Mitochondria
(open arrows) are adjacent to the triad, on the side opposing the Z-line (large black arrow). (B) Mitochondria associated with
the SR terminal cisternae are on the side opposite to that of the Ca2+release channels, or RyRs (arrowheads). The dashed
line shows the shortest distance between release sites (RyR-feet) and the outer mitochondrial membrane. (C–E) Under
higher magnification, small electron-dense strands (black arrows) appear to tether, or bridge, individual mitochondria (Mit) to
the triad. This structural linkage occurs specifically between the mitochondrial outer membrane and the SR terminal cister-
na, generally on the side opposite to that facing the T-tubule (D and E), but also between mitochondria and the lateral sacks
and tubules of the SR that surround the mitochondrion (C). Bars, (A) 0.25 m; (B and C) 0.1 m; (D and E) 0.05 m (Copyright©
2009 by The American Society for Cell Biology with permission from Boncompagniet al.1).
Figure 3. Electron micrographs illustrating the two mito-
chondrial populations in mice gastrocnemius muscle. Black
arrows indicate mitochondria: subsarcolemmal (SS) and in-
termyofibrillar (IMF) mitochondria (×50,000 magnification,
Courtesy of Professor David Hood, University of York).
Muscles, Ligaments and Tendons Journal 2013; 3 (4): 254-266258
E. Barbieri et al.
Molecular and biochemical methods
Mitochondrial life cycle signaling
Most of the molecular studies on muscle mitochondria
have relied on nuclear control of mitochondrial func-
tion. This approach has yielded important findings
over the last few decades with the discovery of multi-
ple transcription factors and cofactors governing mito-
chondrial dynamics, biogenesis and crosstalk with the
nucleus. Furthermore, mitochondria are integral to
other components of stress response or quality control
including ROS signaling, unfolded protein response
and mitochondrial autophagy. The main targets of mi-
tochondrial signaling controlling the biogenesis, dy-
namicity and mitophagy of these organelles in muscle
are summarized in Figure 4.
The mitochondrial life cycle starts with biogenesis,
then mitochondria, through repeated cycles of fission
and fusion, organize themselves into two distinct pos-
sible conditions: discrete units and networks. Finally,
mitochondria life cycle ends with degradation of im-
paired or surplus organelles by mitophagy (turnover),
which involves different biological mechanisms such
as mitoptosis, apoptosis and/or autophagy, depending
on the physio-pathological conditions21,22. In particu-
lar, the mitochondrial biogenesis signaling activated
by peroxisome proliferator-activated receptor gamma
coactivator alpha 1 (PGC-1) members family involves
the transcription factors that regulate the expression
of nuclear genes such as nuclear respiratory factor
(NRF)-1/2 and estrogen-related receptor-α (ERR-α).
These three latter genes control the expression of nu-
clear genes encoding mitochondrial proteins and in-
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Figure 4. Mitohormesis and principal signaling pathways activated in skeletal muscle for mitochondrial biogenesis, remodel-
ing, mitophagy and metabolic activation. Low levels of ROS activate mitochondrial biogenesis and mitochondrial remodeling
through the action of the key regulators PGC-1α , NRF-1, T-FAM, mTFB-1/2, Mitofusin-1/2 and OPA-1. As well as, antioxi-
dant enzymes that function as back-regulators of intracellular ROS levels. These molecules activate cellular mechanisms for
muscle adaptation promoting oxidative metabolism and endogen antioxidants which may prevent oxidative damage and po-
tential chronic diseases. Conversely, high levels of ROS determine mitochondrial damage that leads to fission (DRP-1 and
FIS-1) and subsequent mitophagy (PINK-1, PARKIN and BNIP-3), mitoptosis or even cell death, when removal of impaired
mitochondria together with antioxidant mechanisms are not sufficient to counteract increasing ROS exposure myopathies
and aging related diseases develop.
It is challenging to clearly define the ROS dose required to promote the differing physio-pathological responses, the limits
between the double face of ROS actions is still not clear.
BNIP-3, Bcl-2/adenovirus E1B 19-kDa interacting protein 3; DRP-1, dynamin-related protein 1; FIS-1, mitochondrial fission
protein 1; m-TFB, mitochondrial transcription factor B; NRF, nuclear respiratory factor; OPA-1, optic atrophy 1; p62, polyu-
biquitin binding protein; PGC-1α, peroxisome proliferator-activated receptor gamma coactivator 1 alpha; PINK-1, PTEN-in-
duced putative kinase 1; ROS, reactive oxygen species; T-FAM, mitochondrial transcription factor A.
Muscles, Ligaments and Tendons Journal 2013; 3 (4): 254-266 259
Mitohormesis in muscle cells: a morphological, molecular, and proteomic approach
duce expression of mitochondrial transcription factor A
(T-FAM) and B 1/2 (mTFB), which regulates mtDNA
replication and transcription, thus activating the coor-
dinated expression of mitochondrial proteins14.
Mitochondrial fusion and division are modulated by
GTP-hydrolyzing proteins (GTPases) belonging to the
dynamin superfamily GTPases: mitofusins 1/2 control
outer mitochondrial membrane fusion, optic atrophy 1
(OPA-1) mediates inner mitochondrial membrane fu-
sion and dynamin-related protein 1 (DRP-1) regulate
the division of outer and inner mitochondrial mem-
branes23.
Recent work provides evidence for specific targets that
control the mitochondrial turnover in the course of au-
tophagy-dependent degradation of damaged or depo-
larized mitochondria such as: PTEN-induced putative
kinase 1 (PINK-1), a protein that stabilizes mitochondri-
al integrity and function, important for the maintenance
of mitochondrial networks24; Parkin, which is selectively
recruited from the cytoplasm to damaged mitochondria
by PINK-1 and senses damaged mitochondria for com-
pensatory clearance by autophagy, is25 also involved26.
In addition, Bcl-2/adenovirus E1B 19-kDa interacting
protein 3 (BNIP-3) is a proapoptotic member of the Bcl-
2 family that seems to participate directly in mitophagy
inducing the mitochondrial translocation of DRP-1. No-
tably, DRP-1-mediated mitochondrial fission is correlat-
ed with increased autophagy27. Other target genes are
involved in non selective autophagy, as Atrogins and
the polyubiquitin binding protein p6222.
When compromised by various kinds of damage, soli-
tary mitochondria undergo mitophagy, a self-regulated
eating process that plays key roles in multiple muscle
cell activities. Interplay between mitochondria and au-
tophagy seems to be evolutionarily conserved28. De-
fects in autophagic degradation selective for mito-
chondria are associated with neuromuscular diseases,
emphasizing their physiological relevance to cellular
functions. In response to mitophagy mitochondria
fragment. This morphological change is accomplished
by molecular signals, resulting in the capture of the or-
ganelle by the autophagosomes. By contrast, during
macroautophagy mitochondria fuse in order to avoid
degradation and to sustain ATP production in times of
limited nutrient availability25. The dynamin-related
GTP ases OPA-1 of the inner mitochondrial mem-
brane, and mitofusin1 and 2 of the outer membrane,
control mitochondrial fusion in mammalian muscle
cells. Fission of mitochondria is controlled by DRP-1.
Translocation of DRP-1 from the cytoplasm to mito-
chondria is a crucial step for fragmentation of the or-
ganelle and depends on the Ser 637 residue dephos-
phorylation by calcineurin. By contrast, Ser637 phos-
phorylation by PKA induces mitochondrial elongation.
DRP-1 inside the mitochondria can be stabilized by
SUMO ylation mediated by SUMO ligases, which ex-
plains how dynamic regulation of fission may adapt
mitochondrial morphology to cellular demands25. A
growing body of evidence suggests that autophagy of
mitochondria is a selective and defense-oriented re-
sponse against ROS, mitochondrial dysfunction, and
the accumulation of somatic mutations of mtDNA as-
sociated with aging29; damaged mitochondria are re-
moved by mitophagy by BNIP-3 and mitochondrial fis-
sion protein 1 (FIS-1), a pro-fission mitochondrial pro-
tein that induces mitochondrial fragmentation and en-
hances the extent of mitophagy. Notably, inhibition/al-
teration of mitophagy can contribute to myofiber de-
generation and weakness in muscle disorders charac-
terized by accumulation of abnormal mitochondria and
inclusions30.
A different mechanism by which cells may eliminate
damaged mitochondria is mitoptosis. In the case of a
drop in cellular energy in which mitochondrial stress
leads to impaired oxygen utilization, ROS generation
increases causing mitochondrial network fragmenta-
tion, clustering of damaged mitochondria in the perin-
uclear region, incorporation into a single-membraned-
mitoptotic body, and finally extrusion of the mitoptotic
body via exocytosis or blebbing31. These data are in
agreement with the recent evidence that C2C12 cells
can release in the extracellular space microvesicles
carrying mt DNA32. To date, it is not clear whether this
path is independent or synergic with autophagy.
Lyamzaev et al. discovered that mature mitoptotic
bodies were not associated with autophagosomes,
suggesting that in the setting of whole-cell energy cat-
astrophe, mitoptosis could be more of a parsimonious
mechanism for mitochondrial clearing than mi-
tophagy31. Once damaged organelles are eliminated,
a new mitochondrial pool needs to be generated by
mitochondrial biogenesis.
Mitochondriogenesis may be initiated at the same
time as autophagy or secondary to subsequent au-
tophagic signaling. A number of signals such as
ROS, calcium, energy status, and others influence
the activation of the nuclear coactivator PGC-1α and
the associated nuclear respiratory factors33. The
PGC-1α transcriptional coactivator is a major regula-
tor of energy metabolism. The latter effects of PGC-
1α are likely to represent a compensatory response
where it plays a central role in the adaptation of cellu-
lar energy metabolism and mitochondrial biogenesis.
Hence, mitochondrial homeostasis is reestablished,
and the cell is able to avoid bioenergetic failure and
cell death. With respect to this issue, building on our
group’s previous research15, we have recently ad-
dressed the problem of the role of PGC-1α in C2C12
myoblasts subjected to oxidative stress during the
early stages of differentiation. In particular, we exam-
ined the effect of a mildly toxic concentrations of ex-
ogenously added H2O2 (0.3 mM) on the regulation of
PGC-1α expression and its relationship with AMP-ac-
tivated protein kinase (AMPK) activation (unpublished
observations). In agreement with Kang34 and Ir-
rcher35, we found that 1h treatment with H2O2
markedly increased PGC-1α mRNA expression. It is
noteworthy that, concurrently, we also found an in-
creased phosphorylation of AMPK compared to un-
treated cells, suggesting that oxidative stress induces
PGC-1α through the AMPK signaling pathway. How-
ever, despite the fact that challenged C2C12 my-
Muscles, Ligaments and Tendons Journal 2013; 3 (4): 254-266260
E. Barbieri et al.
oblasts rapidly activate a defense-oriented signaling
cascade, they displayed a 30-40% reduction in their
viability as well as a survivors’ reduced differentiative
efficiency during the post-challenge incubation stage
(up to 7 days of culture). This observation implies
that, in addition to probably being an obligatory and
physiological response to ROS, activation of AMPK
and of PGC-1α may not be sufficient to afford a com-
plete protection to cells against overwhelming oxida-
tive stress. Thus increased mitochondrial production
of ROS is involved at multiple levels in promoting
apoptosis in skeletal muscle cells, an event which is
part of the etiology and progression of numerous
pathologies including sarcopenia and muscle disuse
atrophy, as well as aging36. Romanello et al.37 provid-
ed direct evidence of the importance of the existence
of mitochondrial fission as an amplifying loop in atro-
phying muscles. The mitochondrial network fragmen-
tation induces energy unbalance, which activates a
FoxO3-dependent atrophy program through the
AMPK pathway. Thus, mitochondria play a crucial
role in catabolic muscle signaling: the mitochondrial
fragmentation activates the AMPK-FoxO3 axis, which
induces expression of atrophy-related genes, protein
breakdown and muscle loss.
Mitochondria are about cellular life and death, as
Apostolova N, Blas-Garcia A, Esplugues JV suggest-
ed38, they are directly involved in triggering different
and complexly interconnected programs promoting
cell survival or death. In muscle cells mitochondria
are involved in apoptosis because they contain sever-
al proapoptotic proteins that can lead to cell death
upon release into the cytosol. Chronic muscle disuse
induced by denervation, bed rest, sedentary life style,
aging and mitochondrial impairment leads to an in-
creased susceptibility to mitochondrial mediated
apoptosis, whereas exercise may reduce mitochon-
drially mediated cell death39. The mitochondrial elec-
tron transport chain is the primary source of reactive
oxygen species within the cell. ROS are involved in
apoptosis because they change the conformation of
mitochondrial permeability transition pore (mtPTP)
components to facilitate the release of proapoptotic
proteins and induce cytochrome c (c) release from
the inner membrane and/or the mitochondrial apopto-
sis-inducing channel (MAC). Proapoptotic proteins re-
leased from SS or IMF mitochondria can initiate ei-
ther a caspase-dependent or caspase-independent
cell death pathway.
Mitochondrial isolation method
Most of the molecular and biochemical studies of
muscle mitochondria have been performed on isolat-
ed organelles. Numerous isolation and fractionation
procedures applied to mitochondria as well as their
membrane-bound sub-compartments together with
the identification of specific markers have been re-
ported. One of the major problems in the study of hu-
man muscle mitochondria is the difficulty in obtaining
sufficient amount of fresh tissue, particularly from
healthy human controls. Isolated SSM and IFM mito-
chondria have characteristic lamelliform and tubular
cristae, respectively. The interdependence of mito-
chondrial morphology and function, demonstrated
using isolation methods (the gold standard for the
study of mitochondrial function) force us to reconsid-
er the functional characteristics of isolated or-
ganelles. Isolated mitochondrial preparations from
skeletal muscle cells might present several limiting
factors in experimental preparations. Recent evi-
dence shows that the fragmented mitochondrial
shapes resulting from routine mitochondrial isolation
procedures alters key indices of function in a similar
way to that which occurs when mitochondria undergo
fission in vivo40. Mitochondria can be efficiently iso-
lated by centrifugation and several other techniques,
such as free flow electrophoresis41. Our experience
in isolating mitochondria is specific to cultured mus-
cle cells14. About 3.0x107 C2C12 of myoblasts or
myotubes have to be harvested and washed with 1 ×
PBS buffer. In the case of tissues, about 1gr of tis-
sue is necessary; both samples need to be homoge-
nized. The pellet can be re-suspended in 5 mL of an
ice-cold solution containing 5 mM K-Hepes, pH 7.4,
210 mM mannitol, 1 mM EGTA, 70 mM sucrose, and
55ug/mL digitonin and homogenized by 10 strokes in
an ice-cold glass homogenizer. Non-lysed cells and
nuclei were pelleted by centrifugation at 600-800 g
for 20 min at 4°C, and the supernatant was cen-
trifuged again at 10,000-25,000 g for 15 min at 4°C.
The resulting mitochondrial pellet was re-suspended
in lysis buffer for further biochemical analyses. How-
ever, the mitochondrial fractions contain several con-
taminations from subcellular organelles, such as en-
doplasmic reticulum and lysosomes. To diminish
contaminants, mitochondria may be further purified
using density gradient centrifugation with sucrose,
nycodenz, percoll or metrizamide42.
Proteomic mapping of mitochondria
Proteomics is an attractive tool for the study of mito-
chondria since it allows us to compare the protein ex-
pression profile and protein modifications in tissues,
cells or organelles under different conditions. The ba-
sic procedure for comparative mitochondrial pro-
teomics research includes mitochondria isolation,
large-scale separation of mitochondrial proteins and
protein identification by mass spectrometry.
Mitochondrial proteomics research includes gel-based
and gel-free strategies. The standard gel-based strat-
egy consists in two dimensional gel electrophoresis
(2-DE), in which proteins are separated in the first di-
mension (isoelectrofocusing, IEF), according to their
isoelectic point (pI), on immobilized pH gradient strips
and then, in the second dimension, according to mole-
cular weight (Mw) on conventional SDS-PAGE. For
isoelectric focusing, the choice of an optimal protocol
for sample preparation is critical. The native sample
Muscles, Ligaments and Tendons Journal 2013; 3 (4): 254-266 261
Mitohormesis in muscle cells: a morphological, molecular, and proteomic approach
has to be converted into a suitable physicochemical
state for IEF preserving the native charge and Mw of
the proteins. In most cases, the proteins of the sample
under study need to be solubilized, disaggregated, de-
naturated and reduced.
In our studies mitochondria were re-suspended in
urea lyses buffer composed of: 8M urea, 4% CHAPS,
65 mM DTE and 40 mM Tris base and sonicated for 5
s on ice; after centrifugation the proteins in the super-
natant were separated by 2-DE. Proteins separated
by electrophoresis and intended for in gel digestion
can be visualized by using coomassie brilliant blue,
silver nitrate or fluorescent dyes depending on the
amount of protein in the sample. Gel images are ana-
lyzed with bioinformatic tools to find the differentially
expressed protein spots, which are subsequently ex-
cised from the gel and subjected to in-gel digestion
with trypsin. Finally, the obtained peptides are ana-
lyzed by mass spectrometry to identify the excised
protein. The approach described above, though it has
some limitations, also has clear advantages such as
robustness and technical confidence43.
Mitochondrial proteomics applied to muscle cell
differentiation
Mitochondria act as a potential regulator of myogene-
sis. Several studies have demonstrated that mitochon-
dria are involved in regulating myogenic differentiation
possibly by myogenin, c-Myc, and calcineurin tar-
get14,16,44. In fact, when myoblasts are induced to dif-
ferentiate in the presence of mitochondrial inhibitors,
myogenic differentiation is blocked.
In our investigations, we found that myogenesis ap-
pears to be dependent on mitochondrial function and
mitochondrial biogenesis, as indicated by the rapid
increase in mitochondrial mass/volume, mtDNA copy
number, mitochondrial enzyme activities, and mRNA
levels within the first 48 hrs of myoblast differentia-
tion14. These observations are also in agreement
with Kraft45. In this analysis we integrated the mito-
chondrial changes observed by multiple key determi-
nants using proteomic analysis, presenting the first
proteomic profile of mitochondria during the myogen-
esis program. We used a two dimensional elec-
trophoresis, analyzing the differentially expressed
proteins in the mitochondrial proteome profile during
the myogenic process. We found that enzymes in-
volved in oxidative phosphorylation, such as pyru-
vate dehydrogenase, malate dehydrogenase, fu-
maratehydratase, aconitase, and more markedly HB
and 5B ATP synthase subunits, increased linearly
with mitochondrial biogenesis showing a positive cor-
relation (r2 = 0.915). These findings are consistent
with the increase in mitochondrial function and mem-
brane depolarization. Moreover, they are in agree-
ment with the increase of mitochondrial cristae ob-
served and the reliance on aerobic metabolism
rather than glycolytic metabolism, which character-
izes undifferentiated myoblasts. Among the mito-
chondrial proteins differentially expressed, Man-
ganese Superoxide Dismutase (MnSOD) is one of
the notable proteins involved in the crosstalk be-
tween nuclei and mitochondria. It has recently been
described as a cell protection molecule whose role is
the maintenance of myoblast mitochondrial function
and the preservation of potential myoblast stem cell
differentiation46.
In our model, the increment of MnSOD expression is
highlighted during myotube formation (DifD4 – Dif
D7). Voltage dependent anion-selective channel pro-
tein 1 is another protein associated with myoblast dif-
ferentiation. It has been recognized as a key protein
in mitochondria-mediated apoptosis, since it is a tar-
get for the pro- and antiapoptotic Bcl2-family of pro-
teins, and for its function in releasing apoptotic pro-
teins located in the intermembrane space47. Apopto-
sis, which eliminates cells with defects or undergoing
damage during differentiation, is required for normal
skeletal muscle development48,49. The detection of
mitochondrial myogenesis-correlated proteins, known
to play a role in apoptosis, supports the link between
differentiation and this type of cell death50. Our inves-
tigations did not yield any key proteins up or down
regulated during the myogenic process and required
for the regulation of basal levels of mitophagy. This is
an important process that has received growing atten-
tion, especially in the context of muscle development
in skeletal muscle37, but very little is known about the
key proteins involved and the signals required for the
regulation of basal levels of mitophagy.
In a previous work we demonstrated that a mildly toxic
treatment with H2O2 impaired the ability of myoblasts
to differentiate into mature myotubes15, an event that
is considered as pathologically relevant in specific hu-
man pathologies. In order to evaluate the effects of
oxidative stress specifically on mitochondrial protein
expression we compared the 2D maps of mitochon-
dria purified from differentiating C2C12 exposed or not
exposed to H2O2. The oxidant was given during the
early stage of the myogenic process; cells were ex-
posed for 1 h to 0.3 mM H2O2 24 hr after the onset of
differentiation and then cultured for 2 more days in
fresh, H2O2-free differentiation medium. Mitochondria
were collected before treatment (day 1 of differentia-
tion) and after 2 days (third day of differentiation), to-
gether with the control.
The comparison of 2D maps revealed that mitochon-
dria purified from oxidatively injured cells (Fig. 5b), 2
days after treatment, had a profile of protein expres-
sion similar to that observed in mitochondria purified
from myoblasts (Fig. 5a), except for the MnSOD
whose expression was increased by 3.7 times (Spot
12 in Fig. 5c). This suggests that oxidative stress
blocked the differentiation process and stimulated the
over expression of the detoxifying enzyme. A study of
Lee et al.46 focused on the effects of MnSOD on my-
oblast function in cells derived from both young and
old mice, which under- or over-express the enzyme.
They observed that deficient MnSOD myoblasts im-
paired proliferation and differentiation capacities;
Muscles, Ligaments and Tendons Journal 2013; 3 (4): 254-266262
E. Barbieri et al.
while overexpression of MnSOD preserved myoblast
differentiation potential and mitochondrial mass, de-
spite high levels of intracellular H2O2.
Mitochondrial proteomics applied to exercise re-
search
Several researchers have focused their attention on
the characterization of mitochondrial proteins ex-
pressed in diseased and healthy tissues. These stud-
ies have yielded interesting information leading to im-
portant advances in our understanding of mitochondri-
al functions and cognate molecular signaling. It is rec-
ognized that exercise causes physiological adapta-
tions that depend on the duration, frequency, intensity
of the physical activity, and these adaptations ulti-
mately result in increased oxygen consumption51,52.
Since mitochondria are the most important element in
oxidative metabolism, it is not surprising that a large
range of phenotype modulations associated with exer-
cise occurs on mitochondria.
The first observation, that adaptation to regular en-
durance exercise increases skeletal muscle oxida-
tive capacity and enhances mitochondrial function,
was reported several years ago by Holloszy53. In re-
cent years, mitochondria have become the subject of
studies aiming to understand the molecular mecha-
nisms of skeletal muscle plasticity in healthy and
disease states. Proteomic approaches have identi-
fied alterations in mitochondrial protein abundance;
indicating defects in mitochondrial metabolism in the
skeletal muscle of obese, type 2 diabetic (T2D), and
elderly individuals54. To date, relatively l itt le is
known about the global changes in the mitochondrial
proteome following exercise training. Egan et al.55
investigated the remodeling of the mitochondrial pro-
teome from human skeletal muscle in response to
14 consecutive days of endurance exercise training,
using 2-D DIGE. They analyzed mitochondria-en-
riched protein fractions from human vastus lateralis
muscles taken by biopsy from healthy males. The
volunteers, physically inactive for at least six
months, were subjected to a protocol consisting in
cycling for 60 min per session at 80% VO2max for 14
consecutive days. Three biopsies were taken from
each participant: the first before training, the second
and the third after the 7th day and 14th day, respec-
tively. The short-term endurance training determined
an extensive remodeling of the mitochondrial pro-
teome in human skeletal muscle, suggesting in-
creased capacity for ATP supply, oxygen delivery
and antioxidant capacity, and alterations in the mole-
cular regulatory machinery. Several studies have
documented a decline in mitochondrial function with
aging, but the physiological relevance of this decline
is not clear. Ghosh et al.56 examined the mitochondr-
ial proteome (mitochondria were isolated from hu-
man vastus lateralis) in young and old healthy
sedentary subjects to determine whether the de-
creases in mitochondrial function observed in old mi-
tochondria were associated with changes in the con-
tent of mitochondrial proteins. They found that the
content of several electron transport proteins was re-
duced in the older subjects; in addition, the expres-
sion of PGC-1α, NRF-1, and T-FAM also was also
reduced in older subjects. Remarkably, the investi-
gation also found that a 16-week aerobic exercise
program reversed the mitochondrial phenotype (pro-
teome and function) of old mitochondria. This finding
suggests that exercise training induces a mitochon-
drial remodeling, which provides a mechanistic basis
for physical activity in counteracting age related de-
cline in muscle function and inactivity-associated
metabolic disease progression.
It is well established that contracting skeletal muscle
produces free radicals and it is now clear that both
reactive oxygen species and reactive nitrogen
Figure 5. Proteomic mapping from mitochondria purified from confluent C2C12 cultures subjected to 2-DE before (a) or 3
days after switching to differentiation medium (b-c); b: cells exposed for 1 h to 0.3 mM H2O2 24 hr after the beginning of dif-
ferentiation and then cultured for another 2 days in fresh, H2O2-free differentiation medium; c: control cells at 3 days of dif-
ferentiation time. Spot numbering corresponds to proteins differentially expressed during the C2C12 myogenic process and
identified as described in Barbieri et al.14.
Muscles, Ligaments and Tendons Journal 2013; 3 (4): 254-266 263
Mitohormesis in muscle cells: a morphological, molecular, and proteomic approach
species (RNS) play important roles in cell signaling
pathways involved in muscle adaptation to exercise.
However, it is important to observe that excessive
ROS production can negatively influence exercise
performance and can also be detrimental to health in
the long term57. Mitochondria are commonly cited as
the primary source of superoxide production in con-
tracting muscles; however, recent research suggests
that this may not be the case58. In addition to mito-
chondria, in muscle there are other potential cellular
sites for ROS production such as nicotinamide ade-
nine dinucleotide phosphate (NADPH) oxidases (sar-
coplasmic reticulum, sarcolemma and transverse
tubules) and xantine oxidase, as briefly reported
above. The site of ROS production in muscle may
differ between contracting fibers and fibers exposed
to prolonged inactivity; in fact, Kavazis59 reported
that mitochondrial ROS production largely increases
following prolonged periods of inactivity. Further in-
vestigation is needed to understand the actual role of
mitochondria in ROS and RNS production during
muscle contraction.
Exercise is therefore recognized as an important
agent in improving health, and it is also an auxiliary in
numerous medical treatments and therapy. These
findings regarding exercise have led to a better under-
standing of the plasticity of skeletal muscle, mainly
through the modulation of the mitochondrial proteome
providing insights into muscle sensitivity to exercise
stimulus. All of these results clearly show that pheno-
type changes associated with exercise are linked di-
rectly to mitochondrial proteome modulation. This fact
makes mitochondrial research a promising field in
sports and medical science.
Mitohormesis links physical exercise and reactive
oxygen species generation
Physical exercise consists in planned, structured, and
repetitive bodily movement produced by the contrac-
tion of skeletal muscles resulting in a substantial in-
crease in caloric requirement over resting energy ex-
penditure60,61.
Physical exercise and chronic contractile activity
(CCA) induce a number of physiological adaptations
that ameliorate muscle function and exercise perfor-
mance62. Trained muscle undergoes a remodeling
toward a more oxidative phenotype altering the ul-
trastructural and subcellular organization. These
modifications include an increase in muscle capillar-
ity, extended intramyocellular glycogen and lipid
storage and enhanced insulin responsiveness. How-
ever, one of the most important effects of endurance
training is mitochondrial biogenesis with an increase
in mitochondrial content after only a few weeks of
training63,64. Biogenesis may be mediated by meta-
bolic target activation such as AMPK65. The in-
creased mitochondrial content and mitochondrial ef-
ficiency ameliorate the cellular antioxidant defense
capacity and insulin sensitivity66. It is well- known
that physical exercise increases ROS generation in
skeletal muscle cells7. However, the sources of the
generation of ROS have not yet been explained
Jackson et al., 2007. During exercise, the increased
ROS production is mainly due to the high oxygen
consumption that takes place during increased mito-
chondrial activity, although recent studies have
demonstrated7 several non mitochondrial sources
for the generation of ROS within skeletal muscle
such as the plasma membrane and endomembranes
NADP(H) oxidases8. These studies suggest that
there is probably generation of ROS at multiple sub-
cellular locations in response to a multiplicity of me-
chanical and metabolic stimuli in muscle cells. Re-
cently, it has been demonstrated that exercise-in-
duced ROS function as signaling molecules that
contribute to some of the adaptations occurring after
physical exercise, including an increase in endoge-
nous antioxidant defense capacity and insulin sensi-
tivity. This adaptive response to ROS, often called
mitochondrial hormesis or mitohormesis , has
emerged as a novel and key component not only for
physical exercise adaptation but also for caloric re-
striction and insulin/Insulin like Growth Factor 1 sig-
naling67. Mitohormesis links physical exercise and
the consequent formation of reactive oxygen
species to insulin sensitivity and antioxidant defense
as well as caloric restriction. Generating ROS by
chronic physical activity or caloric restriction: i) stim-
ulates mitochondrial signaling to the cell, ii) reduces
glucose metabolism, iii) induces mitochondrial me-
tabolism, iv) increases stress resistance and gener-
ally prolongs lifespan66.
Physical exercise induces ameliorating effects on in-
sulin resistance by increasing mitochondrial reactive
oxygen species generation in skeletal muscle to stim-
ulate expression of PGCs as insulin sensitivity induc-
ers, as well as SOD 1, 2 and glutathione peroxidase,
strategic enzymes of ROS defense. Remarkably, by
blocking exercise-dependent ROS with antioxidant
supplements, the health benefits of physical exercise
are lost; physical exercise fails to promote insulin sen-
sitivity and antioxidant defense in the presence of vita-
min C and vitamin E66.
The evidence is ambiguous in this regard with a num-
ber of findings suggesting that exposure to ROS may
promote insulin resistance, whereas others suggest
that such exposure may have the opposite effect68,69.
Previous works also contain contradictory findings70.
One possible reason for the divergences among the
various studies may stem from the inverse relation be-
tween ROS and insulin sensitivity obtained in models
of continuous exposure to increased ROS levels69,
whereas current findings64 may imitate transient in-
creases in ROS generation during limited periods of
physical exercise.
This evidence suggests that activation of mitochondri-
al metabolism may promote metabolic health and po-
tentially prolong lifespan. Like calorie and/or glucose
(and possibly amino acid) restriction, physical exer-
cise also induces mitochondrial metabolism activation
Muscles, Ligaments and Tendons Journal 2013; 3 (4): 254-266264
E. Barbieri et al.
and transient ROS formation7,58. Interestingly, it has
been demonstrated that supplementation with antioxi-
dants prevents66 the health benefits of physical exer-
cise71. This suggests that physical exercise shares
common metabolic paths with caloric restriction and
glucose restriction increasing mitohormesis and in-
ducing a positive adaptive response that ends with
stress resistance, antioxidant defense and prolonged
life span.
Conclusions
This review has examined the major cytochemical,
molecular biological, and biochemical techniques
used in investigating muscle mitochondria adaptation
in oxidative stress conditions. Molecular and proteom-
ic techniques, which are steadily improving, may pro-
vide us a better understanding of these processes.
Moreover, the use of such technologies may provide
insights into physiology yielding practical benefits
such as the improvement of biotechnological research
related to life style design and physical activity pre-
scription in a wide array of physiological and patho-
physiological conditions in which mitohormesis may
play a central role.
Acknowledgments
The authors wish to thank the Istituto Interuniversitario
di Miologia (IIM) members for suggestions; Prof. Elisa-
betta Falcieri, Dr. Michela Battistelli and Dr. Sara
Salucci, Department of Earth, Life and Environmental
Sciences of the University of Urbino, Italy, for their
kind support; Prof. Timothy Bloom, Centro Linguistico
d’Ateneo of the University of Urbino, Italy, for a critical
reading of the manuscript.
References
1. Boncompagni S, Rossi AE, Micaroni M, et al. Mitochondria
are linked to calcium stores in striated muscle by develop-
mentally regulated tethering structures. Mol Biol Cell 2009;
20(3):1058-1067.
2. De Stefani D, Raffaello A, Teardo E, Szabo I, Rizzuto R.
A forty-kilodalton protein of the inner membrane is the mi-
tochondrial calcium uniporter. Nature 2011; 476(7360):336-
340.
3. Nunnari J, Suomalainen A. Mitochondria: in sickness and
in health. Cell 2012 16; 148(6):1145-1159.
4. Schapira AH. Mitochondrial diseases. Lancet 2012;
379(9828):1825-1834.
5. Sestili P, Paolillo M, Lenzi M, et al. Sulforaphane induces
DNA single strand breaks in cultured human cells. Mutat
Res 2010; 689(1-2):65-73.
6. Muller FL, Liu Y, Van Remmen H. Complex III releases su-
peroxide to both sides of the inner mitochondrial membrane.
J Biol Chem 2004; 279(47):49064-49073.
7. Powers SK, Jackson MJ. Exercise-induced oxidative
stress: cellular mechanisms and impact on muscle force pro-
duction. Physiol 2008; 88(4):1243-1276.
8. Barbieri E, Sestili P. Reactive oxygen species in skeletal
muscle signaling. J Signal Transduct 2012; 2012:982794.
9. Smith MA, Reid MB. Redox modulation of contractile func-
tion in respiratory and limb skeletal muscle. Respir Phys-
iol Neurobiol 2006; 151(2-3):229-241.
10. Marin-Garcia J. Mitochondria and their role in cardiovas-
cular disease. New York: Springer; 2012.
11. Gomes A, Fernandes E, Lima JL. Fluorescence probes used
for detection of reactive oxygen species. J Biochem Bio-
phys Methods 2005; 65(2-3):45-80.
12. Owusu-Ansah E, Yavari A, Banerjee U. A protocol for _in
vivo_ detection of reactive oxygen species. 2008.
13. Luchetti F, Canonico B, Mannello F, et al. Melatonin reduces
early changes in intramitochondrial cardiolipin during
apoptosis in U937 cell line. Toxicol In Vitro 2007; 21(2):293-
301.
14. Barbieri E, Battistelli M, Casadei L, et al. Morphofunction-
al and Biochemical Approaches for Studying Mitochondr-
ial Changes during Myoblasts Differentiation. J Aging Res
2011; 2011:845379.
15. Sestili P, Barbieri E, Martinelli C, et al. Creatine supple-
mentation prevents the inhibition of myogenic differentia-
tion in oxidatively injured C2C12 murine myoblasts. Mol Nutr
Food Res 2009; 53(9):1187-1204.
16. Rochard P, Rodier A, Casas F, et al. Mitochondrial activ-
ity is involved in the regulation of myoblast differentiation
through myogenin expression and activity of myogenic fac-
tors. J Biol Chem 2000; 275(4):2733-2744.
17. Pizzo P, Drago I, Filadi R, Pozzan T. Mitochondrial
Ca(2)(+) homeostasis: mechanism, role, and tissue speci-
ficities. Pflugers Arch 2012; 464(1):3-17.
18. Franzini-Armstrong C. ER-mitochondria communication. How
privileged? Physiology (Bethesda) 2007; 22:261-268.
19. de Brito OM, Scorrano L. Mitofusin 2 tethers endoplasmic
reticulum to mitochondria. Nature 2008; 456(7222): 605-
610.
20. Connor MK, Bezborodova O, Escobar CP, Hood DA. Ef-
fect of contractile activity on protein turnover in skeletal mus-
cle mitochondrial subfractions. J Appl Physiol (1985). 2000;
88(5):1601-1606.
21. Frank M, Duvezin-Caubet S, Koob S, et al. Mitophagy is
triggered by mild oxidative stress in a mitochondrial fission
dependent manner. Biochim Biophys Acta 2012;
1823(12):2297-2310.
22. Schiaffino S, Dyar KA, Ciciliot S, Blaauw B, Sandri M. Mech-
anisms regulating skeletal muscle growth and atrophy. FEBS
J. 2013; 280(17):4294-4314.
23. Hales KG. The machinery of mitochondrial fusion, division,
and distribution, and emerging connections to apoptosis.
Mitochondrion 2004; 4(4):285-308.
24. Dagda RK, Cherra SJ, 3rd, Kulich SM, Tandon A, Park D,
Chu CT. Loss of PINK1 function promotes mitophagy
through effects on oxidative stress and mitochondrial fis-
sion. J Biol Chem 2009; 284(20):13843-13855.
25. Gomes LC, Scorrano L. Mitochondrial morphology in mi-
tophagy and macroautophagy. Biochim Biophys Acta
2013; 1833(1):205-212.
26. Narendra D, Tanaka A, Suen DF, Youle RJ. Parkin is re-
cruited selectively to impaired mitochondria and promotes
their autophagy. J Cell Biol 2008; 183(5):795-803.
27. Zhang J, Ney PA. Role of BNIP3 and NIX in cell death, au-
tophagy, and mitophagy. Cell Death Differ 2009; 16(7):939-
946.
28. Okamoto K, Kondo-Okamoto N. Mitochondria and au-
tophagy: critical interplay between the two homeostats.
Biochim Biophys Acta 2012; 1820(5):595-600.
29. Mammucari C, Rizzuto R. Signaling pathways in mito-
chondrial dysfunction and aging. Mech Ageing Dev. 2010;
131(7-8):536-543.
Muscles, Ligaments and Tendons Journal 2013; 3 (4): 254-266 265
Mitohormesis in muscle cells: a morphological, molecular, and proteomic approach
30. Masiero E, Agatea L, Mammucari C, et al. Autophagy is re-
quired to maintain muscle mass. Cell Metab 2009;
10(6):507-515.
31. Lyamzaev KG, Nepryakhina OK, Saprunova VB, et al. Nov-
el mechanism of elimination of malfunctioning mitochondria
(mitoptosis): formation of mitoptotic bodies and extrusion
of mitochondrial material from the cell. Biochim Biophys Acta
2008; 1777(7-8):817-825.
32. Guescini M, Guidolin D, Vallorani L, et al. C2C12 myoblasts
release micro-vesicles containing mtDNA and proteins in-
volved in signal transduction. Exp Cell Res 2010;
316(12):1977-1984.
33. Kelly DP, Scarpulla RC. Transcriptional regulatory circuits
controlling mitochondrial biogenesis and function. Genes
Dev 2004; 18(4):357-368.
34. Kang C, O’Moore KM, Dickman JR, Ji LL. Exercise activation
of muscle peroxisome proliferator-activated receptor-gam-
ma coactivator-1alpha signaling is redox sensitive. Free
Radic Biol Med 2009; 47(10):1394-1400.
35. Irrcher I, Ljubicic V, Hood DA. Interactions between ROS
and AMP kinase activity in the regulation of PGC-1alpha
transcription in skeletal muscle cells. Am J Physiol Cell Phys-
iol 2009; 296(1):C116-123.
36. Musaro A, Fulle S, Fano G. Oxidative stress and muscle
homeostasis. Curr Opin Clin Nutr Metab Care 2010;
13(3):236-242.
37. Romanello V, Guadagnin E, Gomes L, et al. Mitochondri-
al fission and remodelling contributes to muscle atrophy.
EMBO J 2010; 29(10):1774-1785.
38. Apostolova N, Blas-Garcia A, Esplugues JV. Mitochondria
sentencing about cellular life and death: a matter of oxidative
stress. Curr Pharm Des 2011; 17(36):4047-4060.
39. Adhihetty PJ, O’Leary MF, Hood DA. Mitochondria in skele-
tal muscle: adaptable rheostats of apoptotic susceptibility.
Exerc Sport Sci Rev 2008; 36(3):116-121.
40. Picard M, Taivassalo T, Gouspillou G, Hepple RT. Mito-
chondria: isolation, structure and function. J Physiol 2011;
589(Pt 18):4413-4421.
41. Zischka H, Weber G, Weber PJ, et al. Improved proteome
analysis of Saccharomyces cerevisiae mitochondria by free-
flow electrophoresis. Proteomics 2003; 3(6):906-916.
42. Chen X, Wei S, Yang F. Mitochondria in the pathogenesis
of diabetes: a proteomic view. Protein Cell 2012; 3(9):648-
660.
43. Rabilloud T, Chevallet M, Luche S, Lelong C. Two-di-
mensional gel electrophoresis in proteomics: Past, present
and future. J Proteomics 2010; 73(11):2064-2077.
44. Seyer P, Grandemange S, Busson M, et al. Mitochondri-
al activity regulates myoblast differentiation by control of
c-Myc expression. J Cell Physiol 2006; 207(1):75-86.
45. Kraft CS, LeMoine CM, Lyons CN, Michaud D, Mueller CR,
Moyes CD. Control of mitochondrial biogenesis during myo-
genesis. Am J Physiol Cell Physiol 2006; 290(4):C1119-
1127.
46. Lee S, Van Remmen H, Csete M. Sod2 overexpression pre-
serves myoblast mitochondrial mass and function, but not
muscle mass with aging. Aging Cell 2009; 8(3):296-310.
47. Shoshan-Barmatz V, Israelson A, Brdiczka D, Sheu SS. The
voltage-dependent anion channel (VDAC): function in in-
tracellular signalling, cell life and cell death. Curr Pharm Des
2006; 12(18):2249-2270.
48. Burattini S, Battistelli M, Falcieri E. Morpho-functional fea-
tures of in-vitro cell death induced by physical agents. Curr
Pharm Des 2010; 16(12):1376-1386.
49. Sandri M, Carraro U. Apoptosis of skeletal muscles during
development and disease. Int J Biochem Cell Biol 1999;
31(12):1373-1390.
50. Casadei L, Vallorani L, Gioacchini AM, et al. Proteomics-
based investigation in C2C12 myoblast differentiation. Eur
J Histochem 2009; 53(4):261-268.
51. Guescini M, Fatone C, Stocchi L, et al. Fine needle aspi-
ration coupled with real-time PCR: a painless methodolo-
gy to study adaptive functional changes in skeletal muscle.
Nutr Metab Cardiovasc Dis 2007; 17(5):383-393.
52. Hawley JA. Molecular responses to strength and en-
durance training: are they incompatible? Appl Physiol Nutr
Metab 2009; 34(3):355-361.
53. Holloszy JO. Biochemical adaptations in muscle. Effects of
exercise on mitochondrial oxygen uptake and respiratory
enzyme activity in skeletal muscle. J Biol Chem 1967;
242(9):2278-2282.
54. Hwang H, Bowen BP, Lefort N, et al. Proteomics analysis
of human skeletal muscle reveals novel abnormalities in obe-
sity and type 2 diabetes. Diabetes 2010; 59(1):33-42.
55. Egan B, Dowling P, O’Connor PL, et al. 2-D DIGE analy-
sis of the mitochondrial proteome from human skeletal mus-
cle reveals time course-dependent remodelling in re-
sponse to 14 consecutive days of endurance exercise train-
ing. Proteomics 2011; 11(8):1413-1428.
56. Ghosh S, Lertwattanarak R, Lefort N, et al. Reduction in
reactive oxygen species production by mitochondria from
elderly subjects with normal and impaired glucose tolerance.
Diabetes 2011; 60(8):2051-2060.
57. Sahlin K, Shabalina IG, Mattsson CM, et al. Ultraendurance
exercise increases the production of reactive oxygen
species in isolated mitochondria from human skeletal
muscle. J Appl Physiol (1985). 2010; 108(4):780-787.
58. Powers SK, Talbert EE, Adhihetty PJ. Reactive oxygen and
nitrogen species as intracellular signals in skeletal muscle.
J Physiol 2011; 589(Pt 9):2129-2138.
59. Kavazis AN, Talbert EE, Smuder AJ, Hudson MB, Nelson
WB, Powers SK. Mechanical ventilation induces di-
aphragmatic mitochondrial dysfunction and increased ox-
idant production. Free Radic Biol Med 2009; 46(6):842-850.
60. Caspersen CJ, Powell KE, Christenson GM. Physical ac-
tivity, exercise, and physical fitness: definitions and dis-
tinctions for health-related research. Public Health Rep 1985;
100(2):126-131.
61. The President Council on Physical Fitness and Sports. De-
finitions - Health, Fitness, and Physical Activity. Washington
(DC)2000. Available from: http://purl.access.gpo.gov/
GPO/LPS21074.
62. Fitts RH. Effects of regular exercise training on skeletal mus-
cle contractile function. Am J Phys Med Rehabil 2003;
82(4):320-331.
63. Adhihetty PJ, Irrcher I, Joseph AM, Ljubicic V, Hood DA.
Plasticity of skeletal muscle mitochondria in response to con-
tractile activity. Exp Physiol 2003; 88(1):99-107.
64. Befroy DE, Petersen KF, Dufour S, Mason GF, Rothman
DL, Shulman GI. Increased substrate oxidation and mito-
chondrial uncoupling in skeletal muscle of endurance-trained
individuals. Proc Natl Acad Sci U S A 2008; 105(43):16701-
16706.
65. Zong H, Ren JM, Young LH, et al. AMP kinase is required
for mitochondrial biogenesis in skeletal muscle in re-
sponse to chronic energy deprivation. Proc Natl Acad Sci
U S A 2002; 99(25):15983-15987.
66. Ristow M, Zarse K, Oberbach A, et al. Antioxidants prevent
health-promoting effects of physical exercise in humans.
Proc Natl Acad Sci U S A 2009; 106(21):8665-8670.
67. Ristow M, Zarse K. How increased oxidative stress promotes
longevity and metabolic health: The concept of mitochon-
drial hormesis (mitohormesis). Exp Gerontol 2010;
45(6):410-418.
68. Goldstein BJ, Mahadev K, Wu X. Redox paradox: insulin
action is facilitated by insulin-stimulated reactive oxygen
Muscles, Ligaments and Tendons Journal 2013; 3 (4): 254-266266
E. Barbieri et al.
species with multiple potential signaling targets. Diabetes
2005; 54(2):311-321.
69. Houstis N, Rosen ED, Lander ES. Reactive oxygen
species have a causal role in multiple forms of insulin re-
sistance. Nature 2006; 440(7086):944-948.
70. Schulz TJ, Zarse K, Voigt A, Urban N, Birringer M, Ristow
M. Glucose restriction extends Caenorhabditis elegans life
span by inducing mitochondrial respiration and increasing
oxidative stress. Cell Metab 2007; 6(4):280-293.
71. Lanza IR, Short DK, Short KR, et al. Endurance exercise
as a countermeasure for aging. Diabetes 2008; 57(11):2933-
2942.
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